Motile properties and localization of contractile proteins in the spermatozoon of discoglossus pictus

Ejaculated spermatozoa from Discoglossus pictus are grouped in bundles, where they are embedded in a network of interwoven threads. Their heads and tails move when exposed to the outermost egg jelly layer, when the bundles are spread across a glass slide or a poly-L-lysine-coated polystyrene tissue culture dish. While moving, single spermatozoa emerge from the bundle and eventually become able to fuse with egg. Spermatozoa were studied by means of immunofluorescent staining, using specific antibodies against actin, myosin, and tubulin, and by means of electron microscopy. Antiactin antibodies stain both the heads and the ridge (which contains the axoneme) of the tail's undulating membrane. Antimyosin antibodies stain the head segment covered by the acrosome cap, and antitubulin antibodies stain the axoneme. The presence of actin and myosin in the spermatozoon head may provide the molecular basis for a cytoskeletal and/or a contractile system which could be involved in the motion sequence as well as subsequent steps of fertilization.     

Protein synthesis in enuleated fertilized and unfertilized mouse eggs

Summary Fertilized and unfertilized C57BL/6J eggs were microsurgically enucleated and then analyzed for their capacity to synthesize proteins using 2-dimensional polyacrylamide gel electrophoresis. In both types of enucleated eggs (cytoplasts), protein synthesis continued and was still detected up to three days in culture. Shortly after enucleation, the pattern of polypeptides remained similar to the respective non-operated control eggs but it later became gradually reduced in intensity and complexity. After two days of culture the appearance of some new proteins typical for 2-cell embryos was observed in enucleated fertilized eggs only. Our findings suggest that maternal mRNA stored during oogenesis is utilized during the preimplantation period.     

Development of the olfactory and vomeronasal organs in Discoglossus pictus (Discoglossidae,Anura)

Using histological techniques and computer‐aided three‐dimensional reconstructions of histological serial sections, we studied the development of the olfactory and vomeronasal organs in the discoglossid frog Discoglossus pictus. The olfactory epithelium in larval D. pictus represents one continuous unit of tissue not divided into two separate portions. However, a small pouch of olfactory epithelium (the “ventromedial diverticulum”) is embedded into the roof of the buccal cavity, anteromedial to the internal naris. The lateral appendix is present in D. pictus through the entire larval period and disappears during the onset of metamorphosis. The disappearance of the lateral appendix at this time suggests that it is a typical larval organ related to aquatic life. The vomeronasal organ develops during hindlimb development, which is comparatively late for anurans. The development of the vomeronasal organ in D. pictus follows the same general developmental pattern recognized for neobatrachians. As with most anurans, the vomeronasal glands appear later than the vomeronasal organ. After metamorphosis, the olfactory organ of adult D. pictus is composed of a series of three interconnected chambers: the cavum principale, cavum medium, and cavum inferius. We suggest that the ventromedial diverticulum at the anterior border of the internal naris of larval D. pictus might be homologous with the ventral olfactory epithelium of bufonids and with the similar diverticulum of Alytes. J. Morphol. 2013. © 2012 Wiley Periodicals, Inc.     

Free intracellular cations in echinoderm oocytes and eggs

The concentrations of Ca²⁺, Na⁺ and H⁺ in echinoderm oocytes and eggs were measured during maturation and activation using ion-selective microelectrodes. In both oocytes and eggs, from three species of starfish and two species of sea urchin, the resting level of cytosolic Ca²⁺ was about 10^-7 M. We did not detect any change in Ca²⁺ concentration either during hormone-induced oocyte maturation (starfish) or during egg activation (starfish and sea urchin) induced by spermatozoa or chemical agents. During 1-methyl-adenine induced maturation of starfish oocytes the intracellular level of Na⁺ increased from 12–35 mM to 40–90 mM, while the pH changed from 6.6–6.8 to 7.0–7.2 Aged oocytes, with intact germinal vesicles, also had elevated levels of Na⁺ and pH.     

Differential expression of alloantigens of the major histocompatibility complex on unfertilized and fertilized mouse eggs

Mouse oocytes at the dictyate and metaphase II stages as well as fertilized eggs have been studied by indirect immunofluorescence for the expression of H-2 histocompatibility antigens on surface membranes. Serologically specific reactivity to H-2 antibody was observed as patchy fluorescence distributed over the surface of the oocyte membrane. In contrast, one-cell zygotes exhibited variable reactivity, and early two-cell stages were negative. Absorption studies confirmed the serologic specificity of the reactivity on oocytes, which could be shown to be due to H-2 antibody. The results suggest that fertilization results in altered expression of major histocompatibility complex surface antigens, and confirms earlier studies that cleavage stage mouse embryos are not reactive with H-2 antibody.     

Development of the ethmoidal structures of the endocranium in Discoglossus pictus (Anura: Discoglossidae)

We use histological techniques and computer‐aided three‐dimensional reconstructions made from serial histological sections to describe the ontogeny of the ethmoidal endocranium of discoglossid frog Discoglossus pictus. We identify a pattern of development for the suprarostral cartilage that differs from previous findings and probably represents the ancestral anuran pattern. The nasal cartilages, including the inferior prenasal cartilage, are de novo adult structures. The only larva‐derived structures of the adult nasal capsules are the posterior aspects of the solum nasi and septum nasi. We also identify patterns of development for the ethmoid plate and postnasal wall that occur during early in ontogenesis. These patterns are associated with development events during metamorphic climax. The pattern of timing of chondrification of the anterior nasal cartilages more closely coincides with that of the neobatrachian species than that recorded for the pelobatid frog Spea. In addition, this study supports a sister taxon relationship between Discoglossus and Alytes. J. Morphol. 271:1078‐1093, 2010. © 2010 Wiley‐Liss, Inc.     

Regionalization and lateral diffusion of membrane proteins in unfertilized and fertilized mouse eggs

The unfertilized mouse egg has a round and highly villated main body and a "nipple" that is unvillated and buds off on fertilization to form the second polar body. Fluorescent markers stain the body more intensely than the nipple, which has been assumed to result from surface amplification due to microvilli. Using fluorescence recovery after photobleaching and microfluorescence photometry, we have measured the membrane protein diffusion and concentration on the main body and nipple region of unfertilized and on fertilized CD-1 mouse eggs. Two general membrane protein labels were used: rhodamine-labeled succinylated concanavalin A and trinitrobenzene sulfonate visualized with a rhodamine Fab fragment of a sheep anti-trinitrophenyl. We found that while the diffusion coefficient was the same on the nipple and main body, considerably higher recovery was observed on the nipple for both probes. The ratio of intensity of fluorescence on the nipple to main body was significantly lower for the concanavalin A stain than for the trinitrophenyl stain, indicating that true concentration gradients exist beyond those that result from surface amplification. The effect of fertilization was not general. No effect was observed for the concanavalin A stain for either diffusion coefficient or percent recovery. For the trinitrophenyl stain, percent recovery decreased approximately twofold while diffusion coefficient increased approximately threefold.     

Changes in elemental composition of fertilized and unfertilized northern pike (Esox lucius) eggs incubated in buffer with and without dimethyl sulfoxide: An X-ray microanalysis study

Fertilized and unfertilized eggs from the northern pike (Esox lucius) were incubated 2 hr in buffer with 0 and 10% (v/v) dimethyl sulfoxide and then quickly frozen in the wells of aluminum blocks submerged in liquid nitrogen. Control eggs and ovarian fluid were similarly frozen immediately after collection. The frozen eggs were sectioned, freeze dried, mounted on stubs, and carbon coated. X-ray microanalysis was used to determine changes in element levels and dimethyl sulfoxide (Me₂SO) penetration in the zona radiata, cytoplasm, cortical alveoli, and egg yolk. Unfertilized eggs incubated without Me₂SO showed decreased levels of Na, Cl, and K in the zona radiata; fertilized eggs, incubated without Me₂SO showed decreased levels of Na, P, and Cl in the zona radiata and increased levels of K in the cytoplasm; unfertilized eggs, incubated with 10% Me₂SO showed decreased Na and Cl in the zona radiata, decreased K in the cytoplasm and increased K in the cortical alveoli; fertilized eggs incubated in buffer with 10% Me₂SO showed decreased levels of Na, P, Cl, and K (zona radiata), P, Cl, and K (cytoplasm), Na (yolk), and increased Cl in the yolk (all P<.01). Me₂SO (v/v) levels reached 1.5-3.1% in the zona radiata, 0-3.2% in cytoplasm, 2.3-8.7% in cortical alveoli, and 0-1.6% in the yolk. Unfertilized eggs showed more Me₂SO penetration than fertilized eggs.     

Uptake of deuterium into proteins of fertilized and unfertilized Arbacia eggs suspended in heavy water

When fertilized and unfertilized eggs of Arbacia punctulata are suspended in heavy water, deuterium is incorporated into stable positions in the egg proteins. The rate of incorporation of the isotope is considerably greater in fertilized than in unfertilized eggs, and is accelerated at the time of formation of the blastula. The result of calculation of the maximum deuterium concentration which would be reached on complete turnover indicates that at least one out of every ten stably bound hydrogen atoms of the egg proteins is a deuterium atom. This has been interpreted as evidence that at the time of formation of the sea urchin blastula and in the period of development which follows, synthesis and breakdown are simultaneous processes leading to the redistribution of amino acids among the egg proteins.     

Spatial distribution of abundant proteins in oocytes and fertilized eggs of the Mexican axolotl (Ambystoma mexicanum)

Two-dimensional protein patterns were compared from sections along the longitudinal axis of oocytes and fertilized eggs of the Mexican axolotl (Ambystoma mexicanum). Only a few differences were observed between four different sections through both oocyte and fertilized eggs. A set of proteins (14 out of 120 proteins) were found that reside only in the germinal vesicles (GV) of the fully grown oocyte. Two of these were observed exclusively in the vegetal half, and one in the animal half after GV breakdown, while other proteins were randomly distributed within the fertilized egg. One cytoplasmic protein was present only in the vegetal half of the mature oocyte and became present also in the animal half of the fertilized egg. Additional proteins were observed in all transverse sections of both mature oocyte and fertilized eggs. It is proposed that these proteins are modified rather than newly synthesized proteins.     

Detection of free oxygen and water vapor in fertilized and unfertilized eggs by diode laser spectroscopy—Exploration of diagnostics possibilities

Nonintrusive methods for characterizing food products are of increasing interest related to the greater awareness of food safety issues. Hen eggs are an important part in food consumption in most parts of the world. We have investigated an optical method utilizing tunable diode lasers for monitoring free gas in eggs. We show that oxygen signals, recorded around 760 nm, increase steadily as eggs become older. Further, we investigated fertilized eggs, which show a quite different temporal behavior during the hatching time. The oxygen signal decreases here with time, while water vapor, recorded around 937 nm, shows a steady increase. Conclusions regarding the size of the air cell and the oxygen availability in fertilized eggs are drawn. The technique might be developed for automatic control of egg freshness, as well as for assessing if eggs are fertilized or not.      

Morphological evolution of Ceratophryinae (Anura, Neobatrachia)

Body form is one of the major consequences of development, and diversification of body shapes implies developmental changes among species. In anurans, changes in the timing of developmental events or heterochrony, have been emphasized as a source of variation in the patterns of development that has lead to diverse morphology. Herein, different approaches are used to explore morphological traits in members of the Ceratophryinae (Anura: Leptodactylidae), a group of frogs with some features produced by overdevelopment. Cladistic analyses were conducted in order to distinguish the shared history of Ceratophrys, Chacophrys and Lepidobatrachus and other anurans. From these studies, morphological variation of selected skeletal features in ceratophryines reveals the presence of ancient structures, which have been considered lost in the neobatrachian phylogeny, integrated in particular designs. Thin-plate spline morphometric analyses of skull shapes among ceratophryines describe Lepidobatrachus as the most distinctive shape. Moreover, thin-plate spline morphometric analyses among anurans show divergent skull shapes between ceratophryines and other anurans, reflecting that the skull shapes of ceratophryines are a result of peramorphosis (increase of developmental rates). This study represents the first detailed examination of the role of peramorphosis in a clade of anurans.     

Gene expression profiling of coelomic cells and discovery of immune-related genes in the earthworm,Eisenia andrei,using expressed sequence tags

The coelomic cells of the earthworm consist of leukocytes, chlorogocytes, and coelomocytes, which play an important role in innate immunity reactions. To gain insight into the expression profiles of coelomic cells of the earthworm, Eisenia andrei, we analyzed 1151 expressed sequence tags (ESTs) derived from the cDNA library of the coelomic cells. Among the 1151 ESTs analyzed, 493 ESTs (42.8%) showed a significant similarity to known genes and represented 164 unique genes, of which 93 ESTs were singletons and 71 ESTs manifested as two or more ESTs. From the 164 unique genes sequenced, we found 24 immune-related and cell defense genes. Furthermore, real-time PCR analysis showed that levels of lysenin-related proteins mRNA in coelomic cells of E. andrei were upregulated after the injection of Bacillus subtilis bacteria. This EST data-set would provide a valuable resource for future researches of earthworm immune system.     

Role of regulatory proteins (troponin-tropomyosin) in pathologic states

In vertebrate striated muscle, troponon-tropomyosin is responsible, in part, not only for transducing the effect of calcium on contractile protein activation, but also for inhibiting actin and myosin interaction when calcium is absent. The regulatory troponin (Tn) complex displays several molecular and calcium binding variations in cardiac muscles of different species and undergoes genetic changes with development and in various pathologic states.Extensive reviews on the role of tropomyosin (Tm) and Tn in the regulation of striated muscle contraction have been published describing the molecular mechanisms involved in contractile protein regulation. In our studies, we have found an increase in Mg²⁺ ATPase activity in cardiac myofibrils from dystrophic hamsters and in rats with chronic coronary artery narrowing. The abnormalities in myofibrillar ATPase activity from cardiomyopathic hamsters were largely corrected by recombining the preparations with a TnTm, complex isolated from normal hamsters indicating that the TnTm, may play a major role in altered myocardial function. We have also observed down regulation of Ca²⁺ Mg²⁺ ATPase of myofibrils from hypertrophic guinea pig hearts, myocardial infarcted rats and diabetic-hypertensive rat hearts. In myosin from diabetic rats, this abnormality was substantially corrected by adding troponin-tropomyosin complex from control hearts. All of these disease models are associated with decreased ATPase activities of pure myosin and in the case of rat and hamster models, shifts of myosin, heavy chain from alpha to beta predominate.In summary, there are three main troponin subunit components which might alter myofibrillar function however, very few direct links of molecular alterations in the regulatory proteins to physiologic and pathologic function have been demonstrated so far.     

Phylogeny and genus-level classification of mantellid frogs (Amphibia, Anura)

We propose a novel classification of frogs in the family Mantellidae, based on published phylogenetic information and on a new analysis of molecular data. Our molecular tree for 53 mantellid species is based on 2419 base pairs of the mitochondrial 12S rRNA, 16S rRNA, tRNAVal and cytochrome b genes, and of the nuclear rhodopsin gene. Because the genus Mantidactylus Boulenger sensu lato is confirmed to be paraphyletic with respect to Mantella Boulenger, and is highly diverse in morphology and reproductive biology, we propose to partition Mantidactylus into seven genera by elevating four subgenera to genus rank (Blommersia Dubois, Guibemantis Dubois, Spinomantis Dubois, and Gephyromantis Methuen) and creating two new genera (Boehmantis gen. n. and Wakea gen. n.). In addition, we create the new subgenera Boophis (Sahona) subgen. n., Gephyromantis (Duboimantis) subgen. n., G. (Vatomantis) subgen. n., and Mantidactylus (Maitsomantis) subgen. n. The following species are transferred to Spinomantis, based on their phylogenetic relationships: S. elegans (Guibé) comb. n. (formerly in Mantidactylus subgenus Guibemantis); S. bertini (Guibé) comb. n. and S. guibei (Blommers-Schlösser) comb. n. (both formerly in Mantidactylus subgenus Blommersia); S. microtis (Guibé) comb. n. (formerly in Boophis Tschudi). Within Boophis, the new B. mandraka species group and B. albipunctatus species group are established. Boophis rhodoscelis (Boulenger) is transferred to the B. microtympanum group. The following five species are revalidated: Mantidactylus bellyi Mocquard and M. bourgati Guibé (not junior synonyms of M. curtus (Boulenger)); M. cowanii (Boulenger) (not syn. M. lugubris (Duméril)); M. delormei Angel (not syn. M. brevipalmatus Ahl); Mantella ebenaui (Boettger) (not syn. M. betsileo (Grandidier)). The new classification accounts for recent progress in the understanding of the phylogeny and natural history of these frogs, but it is still tentative for a number of species. Future modifications may be necessary, especially as concerns species now included in Gephyromantis and Spinomantis.Full article published online at: http://www.senckenberg.de/odes/06-11.htm     

Ultrastructural and lectin binding changes during the formation of the animal dimple in oocytes of Discoglossus pictus (Anura)

The numbers of spores, stalk cells, and basal disk cells in fruiting bodies of Dictyostelium discoideum were estimated by direct cell counting. It was found that the ratios of differentiated cells varied with the number of cells in the fruiting body. Hence, this invalidates, in D. discoideum at least, an assumption used in many theories of differentiation that proportions do not vary with size. Simple statistical analysis showed that a semilogarithmic equation could describe the relationship of spore to stalk cell number and spore to basal disk cell number, whereas a double-logarithmic equation described the basal disk and stalk cell number relationship. Studies under different environmental conditions and with different strains suggest that the basic equations describing the relationships are conserved. However, quantitative differences in the proportioning of the cell types have been observed. Previous papers concerning the proportions of D. discoideum are reviewed, and the implications of the results, in regard to theories of differentiation, are analyzed.